The present invention relates to a fusion protein comprising a Caspase domain or a functionally active variant thereof and a ligand binding domain of a nuclear hormone receptor, a nucleic acid coding for the fusion protein, a vector or cell comprising the nucleic acid, a method of producing the fusion protein, a non-human transgenic animal containing the nucleic acid, the use of the fusion protein for ligand-mediated induction of apoptosis of a cell, or for studying the function of a cell, tissue and/or organ or the use of a transgenic organism for studying the function of a cell at various developmental stages or as a disease model, a method for inducing apoptosis of a cell expressing a fusion protein or for identifying a ligand, or a medicament comprising a fusion protein, the nucleic acid, the vector or the cell, particularly for the treatment of cancer or for or after transplantation, particularly as safety mechanism.
One target of genetic and genomic research is focused on the elucidation of function of individual genes within cells and organisms. Many genes are active only in certain cells and thereby contribute to the complex organisation of the mammalian body composed of hundreds of different cell types. At the level of the whole organism not single genes or gene families interact but population of cell types exist and fulfill biological functions.
To investigate these cellular functions experimentally, mutant analysis is a powerful tool. Like genetic mutants that are used to study the function of individual genes and to create models of genetic disease it is desirable to be able to create mutants for specific cell types or populations of cells in order to study their functional role in vivo. This aspect is of particular interest for the creation of animal models of human degenerative diseases that are characterized by the loss of specific cell populations, e.g. the loss of dopaminergic neurons in Parkinson's disease, or to mimic the damage of specific organs like heart or liver.
Furthermore, cells taken from a donor individual or cells grown in in vitro cultures can be transplanted or transferred into a recipient for research or therapeutic purposes. Upon cell transfer it is desirable to be able to ablate specifically all or some of the transplanted cells either to study the functions these cells fulfill in the recipient body or to enhance the safety of cell therapy if the transplanted cells thread the recipient by e.g. tumorigenesis or a graft versus host reaction (Cohen, et al., Immunol Today, 20, 172-176 (1999)) (Cohen, et al., Leuk Lymphoma, 34, 473-480 (1999)) (Cohen, et al., Hum Gene Ther, 10, 2701-2707 (1999) (Berger, et al., Blood, 103, 1261-1269 (2004)).
Moreover, in a cancer therapy termed suicide gene therapy tumor cells are equipped with an expression vector for a gene that allows to destroy these cells upon administration of a specific drug (Hurwitz, et al., Hum Gene Ther, 10, 441-448 (1999)) (Fillat, et al., Curr Gene Ther, 3, 13-26 (2003)) (Niculescu-Duvaz and Springer, Mol Biotechnol, 30, 71-88 (2005)) (Portsmouth, et al., Mol Aspects Med, 28, 4-41 (2007)).
In conclusion, it is an important aspect of biological and medical research to be able to manipulate the cellular composition of an organism such as the mammalian body. Ideally, methods would be available that enable cell ablation in a specific and also in a timed manner and that are safe, simple and universally applicable to all cell types and organs of the mammalian body.
Over the last two decades a variety of genetic methods has been developed to ablate selected cells in the body, mostly using the mouse as a model organism. These strategies can be classified into non-inducible methods that cannot be regulated from outside and lead to preprogrammed cell death during development and into inducible methods that employ initially innocent transgenes that are able mediate cell death upon administration of an inducer molecule.
The various methods are further distinguished by the biochemical mechanisms that lead to cell death, i.e. either by the accumulation of toxic products and necrotic cell death or by the use of endogenous pathways that lead to programmed cell death through apoptosis. The innate immune system reacts differently to cells that underwent pathological (necrosis) or physiological cell death (apoptosis) such that the clearance of necrotic cells is associated with proinflammatory responses of phagocytic macrophages (Cocco and Ucker, Mol Biol Cell, 12, 919-930 (2001)) (Krysko, et al., Apoptosis, 11, 1709-1726 (2006)). Therefore, the latter method is most appropriate to model disease processes that involve apoptotic cell death.
The strategies for non-inducible cell ablation in mice have used transgenes that employ the cell type specific expression of toxic proteins like the diphtheria toxin A chain (Breitman, et al., Science, 238, 1563-1565 (1987)) (Breitman, et al., Mol Cell Biol, 10, 474-479 (1990))(Kaur, et al., Development, 105, 613-619 (1989)) or Ricin (Landel, et al., Genes Dev, 2, 1168-1178 (1988)). This method was later refined such that the expression of diphtheria toxin can be controlled by the activity of Cre recombinase. In such double transgenic mice Cre recombinase is expressed from a cell type specific promoter while the diphtheria toxin transgene is under control of an ubiquitous active promoter but toxin expression occurs only upon Cre mediated deletion of an inhibitory DNA segment (Brockschnieder, et al., Mol Cell Biol, 24, 7636-7642 (2004)) (Brockschnieder, et al., Genesis, 44, 322-327 (2006)) (Ivanova, et al., Genesis, 43, 129-135 (2005)). Non-inducible cell ablation strategies rely solely on the activity of cell type specific promoter region, the activity of which cannot be further influenced in vivo. Thus, cell ablation occurs upon the initial activation of the utilised promoter region during embryonic development.
To gain also control on the timing of cell ablation a variety of inducible ablation strategies has been developed. Two of these methods are based on the transgenic expression of prokaryotic enzymes that modify specific prodrugs into cytotoxic derivates. The prodrugs are not recognised by mammalian enzymes. Thus, the cells expressing the prokaryotic enzyme are only killed upon the administration of the specific prodrug.
The use of a thymidine kinase derived from Herpes simplex virus (HSV-tk) enables to kill HSV-tk expressing, dividing cells by the administration of Ganciclovir (GANC) (Sofroniew, et al., Brain Res, 835, 91-95 (1999)) (Visnjic, et al., J Bone Miner Res, 16, 2222-2231 (2001)) (Rindi, et al., Development, 126, 4149-4156 (1999)) (Tian, et al., Am J Pathol, 163, 789-801 (2003)) (Ito, et al., Nat Med, 11, 1351-1354 (2005)) (Dancer, et al., Gene Ther, 10, 1170-1178 (2003)) (Lalancette-Hebert, et al., J Neurosci, 27, 2596-2605 (2007)) (Zhang, et al., Febs J, 272, 2207-2215 (2005)). GANC is phosphorylated only by HSV-tk and then blocks DNA replication leading to the death of mitotic cells. Postmitotic, resting cells cannot be ablated with the HSV-tk/GANC system.
The use of the Nitroreductase (NTR) gene derived from E. coli enables to kill NTR expressing cells by the administration of the prodrug CB1954 (Clark, et al., Gene Ther, 4, 101-110 (1997)) (Cui, et al., Glia, 34, 272-282 (2001)) (Isles, et al., J Neurobiol, 47, 183-193 (2001)) (Gusterson, et al., Recent Results Cancer Res, 163, 31-45 (2003)). The cytotoxic derivative leads to the formation of interstrand DNA crosslinks which are poorly repaired by the cells. The NTR system is independent of the cell cycle and can be applied to non-dividing cells (Grove, et al., Cancer Res, 63, 5532-5537 (2003)). The prodrug CB1954, however, has evolved from cancer therapy and a significant bystander effect has been observed because of local spread of the activated prodrug that leads to the death of neighbored cells (Bridgewater, et al., Hum Gene Ther, 8, 709-717 (1997)) (Nishihara, et al., Anticancer Res, 18, 1521-1525 (1998)). While this effect is beneficial for cancer therapy it diminishes the utility of the NTR system for specific cell ablation.
In another inducible approach cells that express a receptor for diphtheria toxin (DTR) from a cell type specific transgene can be killed by the in vivo administration of diphtheria toxin A chain (DTA) (Buch, et al., Nat Methods, 2, 419-426 (2005)) (Chang and Yang, Sci STKE, 2003, PL1 (2003)) (Stoneman, et al., Circ Res, (2007)). DTA is toxic upon internalisation that is mediated by the transgenic DTR.
Besides the use of toxins or enzymes that lead to cytotoxic products two methods for inducible cell ablation have been developed that exploit endogenous cellular mechanisms of programmed cell death.
In the system described by Takebayashi (Takebayashi, et al., Cancer Res, 56, 4164-4170 (1996)) the transmembrane and intracellular domain of the Fas death receptor (amino acid 135-305) has been fused N-terminally to the ligand binding domain of the rat estrogen receptor. This fusion protein was constitutively expressed in L929 cells known to be sensitive to Fas-mediated apoptosis. From studies with wildtype estrogen receptor it has been found that upon ligand administration the ER domain undergoes a conformational change that leads to the dissociation of bound heat shock proteins and receptor dimerisation. The administration of estradiol to Fas-ER expressing L929 cells, T-lymphocytes or HeLa cells leads to cell death by apoptosis (Takebayashi, et al., Cancer Res, 56, 4164-4170 (1996)) (Kawaguchi, et al., Cancer Lett, 116, 53-59 (1997)) (Kametaka, et al., Cancer Sci, 94, 639-643 (2003)).
In a variation of this method the non-modified ER domain was replaced by a mutant murine ER ligand binding domain (amino acids 287-599) that harbours a single amino acid exchange (G525R). This mutation leads to a strongly reduced affinity to estradiol but the receptor can still be activated by 4-OH-tamoxifen. This Fas-ER(G525R) fusion protein was tested in the mouse cell line L929 (Kodaira, et al., Jpn J Cancer Res, 89, 741-747 (1998)). The Fas-ER method uses the extrinsic CD95 apoptosis pathway to induce cell death. Since this pathway is restricted in vivo largely to cells of the immune system (Krammer, Nature, 407, 789-795 (2000)) most other cell types in the body may be unresponsive to Fas-ER fusion proteins.
A cell ablation method that utilises ubiquitously expressed components of the intrinsic apoptosis pathways was first described by MacCorkle (MacCorkle, et al., Proc Natl Acad Sci USA, 95, 3655-3660 (1998)). For this method a domain of the FK506 binding protein FKBP was fused to the N-terminus of Caspase-1 or Caspase-3 and expressed in human Jurkat T cell lymphoma cells. Upon administration of dimeric FK506 (FK1012; Pruschy, et al., Chem Biol, 1, 163-172 (1994)), a chemical inducer of dimerisation (CID), the fusion proteins undergo oligomerisation and lead to cell death by apoptosis. This system was further developed by the fusion of one or more modified FKBP domains (Fv) to the N-terminus of Fas, Bax, Caspase-1, -3, -8 and -9 (Fan, et al., Hum Gene Ther, 10, 2273-2285 (1999)) (Hou and Hsu, Am J Physiol Heart Circ Physiol, 289, H477-487 (2005)). The Fv domain can be dimerised by the FK1012 analogs AP1903 (Fan, et al., Hum Gene Ther, 10, 2273-2285 (1999)) or AP20187 (Chang, et al., J Biol Chem, 278, 16466-16469 (2003)) that exhibit a higher affinity to the modified Fv domain than to the wildtype FKBP. However, FK506 and analogs that bind to FKBP exhibit a strong immunosuppressive action in vivo (Bierer, et al., Curr Opin Immunol, 5, 763-773 (1993)). The CID apoptosis system has been used for the ablation of transplanted endothelial cells in vivo that were transduced with a viral vector expressing a Fv-Caspase-9 fusion protein (Nor, et al., Gene Ther, 9, 444-451 (2002)) and to demonstrate suicide gene therapy of prostate cancer cells with a viral vector expressing a Fv-Caspase-1 protein (Shariat, et al., Cancer Res, 61, 2562-2571 (2001)). This system was further used in transgenic mice expressing a Fv-Caspase-3 fusion protein in hepatocytes as a model of inducible liver injury (Mallet, et al., Nat Biotechnol, 20, 1234-1239 (2002)) and in transgenic mice expressing a Fv-Caspase-8 fusion protein in adipocytes to create a model of inducible lipoatrophy (Pajvani, et al., Nat Med, 11, 797-803 (2005)).
Although great efforts have been undertaken to derive systems that allow inducible cell ablation in the mammalian body the existing technologies have severe limitations that limit their practical use:    1. The expression of diphtheria toxin from a cell type specific promoter or the activation of a diphtheria toxin gene through Cre recombinase expressed from a cell type specific promoter does not allow the induction of cell ablation from outside and does not provide control on the timing of cell ablation.    2. The ablation of cells expressing HSV-thymidine kinase by the administration of GANC enables induction from outside but this system is restricted to actively proliferating cells. Resting cells like mature neurons cannot be ablated.    3. The nitroreductase system is derived from cancer therapy and can lead to nonspecific cell death of neighbouring cells.    4. The activation of a diphtheria toxin receptor gene through Cre recombinase expressed from a cell type-specific promoter followed by administration of diphtheria toxin is impractical because it requires two independent transgenes and the generation of double transgenic mice.    5. The utility of the Fas-ER(G525R) fusion protein is restricted only to cells that are responsive to the CD95 extrinsic apoptosis pathway, i.e. mostly cells of the immune system.    6. The inducible CID system in combination with active Caspase domains has been developed for in vitro use and has limitations for in vivo application with respect to the pharmacology of the inducing compounds. The first generation inducer FK1012 (as a dimer of FK506; Pruschy, et al., Chem Biol, 1, 163-172 (1994)), and putatively also the analogs that bind to the endogenous FKPB protein, from which the CID dimeriser domain is derived, are immunosuppressive (Bierer, et al., Curr Opin Immunol, 5, 763-773 (1993)). The in vivo pharmacokinetics, metabolism and toxicity of these compounds (e.g. AP20187; (Chang, et al., J Biol Chem, 278, 16466-16469 (2003)) has not been characterised. Furthermore, it is not known whether any of these compounds penetrates the blood-brain barrier such that the utility of the CID system for use in the brain is unpredictable.